Clinical test agents using an enzyme specifically reacting to a specific substrate are used for measuring various internal molecules, and a representative example thereof is a test agent for glucose measurement.
A test agent for glucose measurement uses the property of an enzyme for glucose determination, for example glucose dehydrogenase (GDH), to catalyze the dehydrogenation reaction of glucose, and can determine the glucose concentration in an analysis sample based on this property.
Although as GDH, NAD(P)+GDH derived from Bacillus megaterium can be also used for example, there is a problem that the thermal stability is extremely low when an inorganic salt does not exist in an excessive amount.
Therefore, an attempt to improve the thermal stability, the pH stability or the specific activity in the absence of an inorganic salt has been made, by producing a mutant of NAD(P)+GDH derived from Bacillus megaterium in which specific amino acids are replaced with other amino acids (for example, Patent Documents 1 to 3 and Non-Patent Documents 1 and 2).
In Patent Document 3, it is described that the mutant of NAD(P)+GDH derived from Bacillus megaterium, in which glutamic acid at residue position 170 is replaced with lysine and glutamine at residue position 252 is replaced with leucine, maintains a relative activity of about 60% even after the treatment at 66° C. for 8 hours in the absence of an inorganic salt.
Regarding NAD(P)+GDH derived from Bacillus subtilis, which is classified into genus Bacillus as Bacillus megaterium above, the enzyme has been isolated and its gene has been already identified (for example, Non-Patent Documents 3 and 4), and the enzyme can be also used for the application as a test agent for glucose measurement.
NAD(P)+GDH derived from Bacillus subtilis above is an enzyme, which shows about 85% homology with NAD(P)+GDH derived from Bacillus megaterium (for example, Non-Patent Document 5), and has a high specific activity of 900 U/mg or more in the presence of sodium chloride with a high concentration.
Its thermal stability in the absence of an inorganic salt and the like, however, was not sufficient as NAD(P)+GDH derived from Bacillus megaterium. Thus, regarding NAD(P)+GDH derived from Bacillus subtilis, an attempt to improve the thermal stability, the pH stability or the relative activity in the absence of an inorganic salt has been also made, by producing a mutant in which specific amino acids are replaced with other amino acids.
On the other hand, a new application of recent GDH is the reproduction of NAD(P)H. When the reaction of NAD(P)+GDH catalyzing glucose is coupled with a reaction system, which uses NAD(P)H and produces NAD(P), expensive NAD(P)H can be sometimes reproduced; however, the thermal stability of NAD(P)+GDH and the like have been a problem also in this case.
Patent Document 4 describes that, in the application for the reproduction of NAD(P)H, a mutant of NAD(P)+GDH derived from Bacillus subtilis, in which at least one amino acid of isoleucine at residue position 165, proline at residue position 194 and lysine at residue position 204 is replaced with another amino acid and other amino acid(s) is also replaced, has an improved specific activity that is several times higher than that of the wild-type enzyme, and has a remaining activity of 80% or more after the heat treatment at 50° C. for 20 minutes.
Non-Patent Document 6 describes that, also in the application for the reproduction of NAD(P)H, the mutant of NAD(P)+GDH derived from Bacillus subtilis, in which proline at residue position 45 is replaced with alanine, phenylalanine at residue position 155 is replaced with tyrosine, glutamic acid at residue position 170 is replaced with arginine, valine at residue position 227 is replaced with alanine and glutamine at residue position 252 is replaced with leucine, and other mutants have thermal stability, in which almost no in activation is observed at 65° C. in the presence of 0.3 M sodium chloride, and have specific activities of 100 to 150 U/mg.
Non-Patent Document 7 describes that, also in the application for the reproduction of NAD(P)H, the mutant of NAD(P)+GDH derived from Bacillus subtilis, in which proline at residue position 45 is replaced with alanine, asparagine at residue position 46 is replaced with glutamic acid, phenylalanine at residue position 155 is replaced with tyrosine, glutamic acid at residue position 170 is replaced with lysine, valine at residue position 227 is replaced with alanine, tryptophan at residue position 230 is replaced with phenylalanine and glutamine at residue position 252 is replaced with leucine, has thermal stability in which almost no deactivation is observed at 65° C., and that its resistance to an organic solvent such as acetone is improved in comparison, with the wild-type enzyme.
Further, another new application of NAD(P)+GDH is a biofuel cell. A biofuel cell, in which an oxidoreductase is immobilized as a catalyst on at least one of the negative electrode and the positive electrode, attracts the attention as a next-generation fuel cell, since it can effectively extract electrons from a fuel, which cannot be used with a normal industrial catalyst such as glucose. As described in Patent Document 5 or Patent Document 6 for example, NAD(P)+GDH is used as an important enzyme to extract electrons from glucose first at the negative electrode.